The present invention relates to the coupling of a liquid-phase analysis technique such as liquid chromatography with Nuclear Magnetic Resonance (xe2x80x9cNMRxe2x80x9d) spectroscopy.
The practice of modern high-resolution NMR spectroscopy on a sample dissolved or otherwise borne in a liquid phase requires that the uniformity of the applied static magnetic field (B0) be maintained to the parts-per-billion level over the actively-interrogated sample region. Considerable effort has been expended over many years to minimize the contribution of sample probe hardware to B0 field non-uniformity. For example, in U.S. Pat. No. 5,684,401, Peck et. al. teach the use of a susceptibility-matching medium to significantly reduce the B0 distortion attributable to differences in the magnetic susceptibilities of the various materials (including air) which comprise, or which reside adjacent to, a microcoil flow cell. Magnetic susceptibility is a measure of the extent to which magnetization can be induced in a material when subjected to an externally applied magnetic field. Each component of the flow cell, as well as the solvent, has associated with it a magnetic susceptibility. The magnetic susceptibility of the solvent stream, which bears the sample, is one of the variables that can contribute to non-uniformity of the field in the immediate vicinity of the sample. Poor B0 field uniformity results in broad and distorted NMR spectral lineshape, as disclosed in the Peck reference and in the literature cited therein.
In cases where a flow-through cell is used, but where there is substantially no change of solvent composition across the active sensing region of the flow cell, it is generally possible to xe2x80x9cshimxe2x80x9d the spectrometer to the extent that good quality NMR spectra are produced. As used, the term xe2x80x9cshimmingxe2x80x9d refers to the superposition of secondary fields for the purpose of locally minimizing the non-uniformity of B0 in the vicinity of the sample. However, in the event that a change of solvent composition is impressed across the probe, as would be the case in gradient elution liquid chromatography, a dramatic reduction in spectral quality can result. Typical liquid chromatography solvents as would be used in reverse phase gradient elution have magnetic susceptibilities which differ at the part-per-million level, whereas the desired B0 uniformity is at the parts-per-billion level. In practice, it becomes apparent that solvent composition differences of relatively small magnitude (from a few per cent to less than one per cent), when impressed across the NMR detector cell volume, can result in demonstrably degraded NMR spectra, which may be rendered unusable for structure elucidation. This phenomenon dramatically diminishes the ability to utilize, for example, gradient-mode liquid chromatography to bring about sample focussing or other modes of sample manipulation, within a liquid inlet system to an NMR spectrometer.
Several approaches to solving the above-referenced problem have been attempted within the prior art. At least one commercial NMR spectrometer manufacturer offers a hardware accessory for use with conventional-scale liquid chromatography, which allows the collection of the HPLC eluent into a series of discrete loops. The contents of the individual loops are analyzed by the NMR spectrometer off-line from the chromatography in a separate sequence of analyses. Solvent compositional variations, which may exist over the length of a sample loop at the time of fluid capture, have an opportunity to diffuse toward equilibrium. At the time of the secondary analysis, the loop contents can be delivered into the NMR detection cell by a solvent of choice, which may be chosen to match the properties of the elution solvent at the time of a given sample""s capture. Additionally, the loop volume and the detection cell volume may be chosen in such a way that the equilibrated loop contents overfill, or fully flush through, the interrogated volume of the detector cell.
There are several significant disadvantages of this approach. Most importantly, the NMR spectrometer is no longer on-line. The NMR analysis requires a separate and distinct set of processes to xe2x80x9cre-playxe2x80x9d the segmented chromatogram. The volume of the individual loops defines and limits the volumetric resolution of the chromatographic separation (i.e. the coarseness of the segmentation). At a defined loop volume, the number of loops available limits the overall volume of the chromatographic separation which is preserved.
If fluid transfer to the loop storage device is being steered xe2x80x9cintelligentlyxe2x80x9d, as by an auxiliary detector such as a UV absorbance detector or a mass spectrometer, in order to selectively capture peaks of interest within a chromatogram, it becomes a requirement that the peaks of interest also be detectable by this alternate detection means. A compound of NMR interest, with no useable UV chromophore, would be undetectable by the UV detector and would therefore by-pass collection in this scheme. Similarly, a compound of NMR interest, for which ionization and analysis conditions were not properly pre-established at the mass spectrometer, would be undetectable by the mass spectrometer, and would also by-pass collection.
Additionally, disadvantageously the loop storage device requires the presence of a switching valve in-line with the chromatographic stream to direct the flow into the target loop. The presence of switching valve hardware can further degrade the fidelity of an eluting zone or band. Such degradation is particularly evident as the volume is reduced from a conventional chromatographic volume scale to a capillary chromatographic volume scale.
Other attempts to minimize the effect of solvent composition and susceptibility variation on NMR spectral quality, within the prior art, have been to limit the on-line operation to use with only extremely shallow compositional gradients. If the user-programmed time-rate-of-change of solvent composition is sufficiently shallow, then the resulting volume-rate-of-change of composition produced can be shallow enough that the compositional difference encompassed by the actively-interrogated volume of the NMR detection cell tends toward insignificance. In the case of an infinitely shallow gradient, there is substantially no compositional difference impressed across the cell, but correspondingly the liquid-phase separation is effectively isocratic (time-invariant).
Restricting LC-NMR operation to shallow composition gradients of typically 1 per cent composition change per minute or less, imposes significant limitations on the user. The analysis time becomes very lengthy if a large compositional range is to be spanned by the gradient. Compositional gradients are commonly employed to permit the separation and analysis of materials of widely differing retention behavior. A typical gradient profile may call for the percentage of a given solvent component to change by 40, 60 or even 100 percent, in order to allow elution of all of the analyte species present. If a large-percentage change is limited to occur at a rate less than or equal to 1 per cent per minute, then the analysis time may become unacceptably long.
Additionally, shallow gradients may not achieve the same sample focussing or concentrating behavior that a steeper gradient can provide. In certain modes of operation, a user may wish to introduce a sample, which resides at a dilute concentration in a relatively large volume of liquid. That sample may be focussed or concentrated on a chromatography column under an initial set of elution conditions where the analyte is strongly retained, and where the liquid in which the sample was initially dissolved is flushed through the column. The user may then apply a relatively steep compositional gradient to elute the material off of the column and into the spectrometer in a very highly concentrated band, in order to optimize detection. In such a mode of operation, the programmed gradient which is employed may have a time-rate-of-change of composition of many per cent per minute. The corresponding volume-rate-of-change of composition may be used to affect the overall volume in which the band elutes. A limitation requiring the use of shallow gradients impairs or inhibits the rapid use of this mode of operation.
Consequently there are numerous limitations associated with prior approaches to solve the problems caused by variation of the magnetic susceptibility of the solvent surrounding the sample subjected to NMR analysis.
The present invention provides a method in which the limitation upon analysis caused by variation of the magnetic susceptibility of the solvent incorporating a sample can be addressed.
According to the invention, solvent composition which is used to bring about elution of an analyte from a device such as a chromatography column, can be varied without causing a corresponding variation of the solvent composition used to transport the analyte to an NMR spectrometer. The analytical stream has summed therewith a solvent composition which is complementary to the instantaneous composition emerging from a liquid-phase separation or analysis, such that the composition of the final output stream remains constant. The method according to the invention, can be accomplished by either an open-loop approach where a time-programmed gradient is executed substantially simultaneously by two solvent gradient generation systems, or by a closed loop approach where the system actively senses a property of the solvent stream arriving at a sensing cell located downstream of the column or other analytical device, and modulates the output of the compensating pumps in order to maintain that measured solvent property substantially constant.
In the open loop method according to the invention, a first solvent gradient is applied to the sample injector and column to bring about a sample elution from the column. A secondary solvent gradient having a complementary composition to the first gradient is sent through a delay volume, which mimics the delay attributable to the presence of the sample injector, column, and optional chromatographic detector in the analytical side of the system. The solvent flow and composition produced by the second gradient generator is summed with the analytical flow in a low-band broadening tee geometry such as the Nano-Tee currently commercialized by Waters Corporation, of Milford Mass. The accuracy with which composition change produced within the analytical side of the system is nulled is a function of the inherent compositional accuracies of the two gradient generators and of the matching of the compositional delay characteristics of the analytical and the compensation flow paths.
In the closed loop method according to the invention, the need for matching the fluid circuit of the chromatographic or analytical path with analogous fluidic components in the compensation or secondary mobile phase delivery path is eliminated. The closed loop method instead actively senses a solvent property such as the instantaneous solvent composition at a sensing cell located downstream of the column. The system controller modulates the output of the compensating pumps in order to maintain the measured solvent property substantially constant.
Features of the invention include provisions that allow for the analysis to be performed on-line. This ability to conduct analysis on line allows analysis to be conducted without a separate and distinct set of processes to replay the segmented chromatogram. Moreover, the volumetric resolution of the chromatographic separation is not limited by a defined loop volume, nor is it necessary to include a switching valve that diminishes the fidelity of the eluting zone or band. Additionally, the analysis is not restricted to shallow gradients.